Chemokine b-4 polypeptide

ABSTRACT

Human chemokine polypeptides and DNA (RNA) encoding such chemokine polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such chemokine polypeptides for the treatment of leukemia, tumors, chronic infections, autoimmune disease, fibrotic disorders, wound healing and psoriasis. Antagonists against such chemokine polypeptides and their use as a therapeutic to treat rheumatoid arthritis, autoimmune and chronic inflammatory and infective diseases, allergic reactions, prostaglandin-independent fever and bone marrow failure are also disclosed. Diagnostic assays for identifying mutations in nucleic acid sequence encoding a polypeptide of the present invention and for detecting altered levels of the polypeptide of the present invention are also disclosed.

[0001] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptides ofthe present invention are human chemokine beta-4 and human chemokinebeta-10, sometimes hereinafter referred to as “Ckβ-4” and “Ckβ-10”,collectively referred to as “the chemokine polypeptides”. The inventionalso relates to inhibiting the action of such polypeptides.

[0002] Chemokines are an emerging super-family of small secretedcytokines that are structurally and functionally related. All chemokinesexhibit 25 to 75% homology at the amino acid level and contain spatiallyconserved cysteine residues as do the polypeptides of the presentinvention. Members of the “C-X-C branch” (according to the position ofthe first two cysteines in the conserved motif), also known asneutrophil-activating peptide (NAP)/IL-8 family, exert pro-inflammatoryactivity mainly through their action on neutrophils (e.g., IL-8 andNAP-2), whereas members of the “C-C branch” family appear to attractcertain mononuclear cells. Members of the “C-C branch” include PF4,MIPs, MCPs, and the chemokine polypeptides of the present invention.

[0003] Numerous biological activities have been assigned to thischemokine family. The macrophage inflammatory protein 1α and 1β arechemotactic for distinct lymphocyte populations and monocytes (Schall,T. J., Cytokine, 3:165 (1991)), while MCP-1 has been described as aspecific monocyte chemo-attractant (Matsushima, K., et al., J. Exp.Med., 169:1485 (1989)). The common function of this chemokine family istheir ability to stimulate chemotactic migration of distinct sets ofcells, for example, immune cells (leukocytes) and fibroblasts. Thesechemokines are also able to activate certain cells in this family.

[0004] The immune cells which are responsive to the chemokines have avast number of in vivo functions and therefore their regulation by suchchemokines is an important area in the treatment of disease.

[0005] For example, eosinophils destroy parasites to lessen parasiticinfection. Eosinophils are also responsible for chronic inflammation inthe airways of the respiratory system. Macrophages are responsible forsuppressing tumor formation in vertebrates. Further, basophils releasehistamine which may play an important role in allergic inflammation.Accordingly, promoting and inhibiting such cells, has wide therapeuticapplication.

[0006] In accordance with one aspect of the present invention, there areprovided novel polypeptides which are Ck-4, and Ckβ-10, as well asfragments, analogs and derivatives thereof. The polypeptides of thepresent invention are of human origin.

[0007] In accordance with another aspect of the present invention, thereare provided polynucleotides (DNA or RNA) which encode suchpolypeptides.

[0008] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptides byrecombinant techniques.

[0009] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides for therapeutic purposes, forexample, to treat solid tumors, chronic infections, auto-immunediseases, psoriasis, asthma, allergy, to regulate hematopoiesis, and topromote wound healing.

[0010] In accordance with yet a further aspect of the present invention,there are provided antibodies against such polypeptides.

[0011] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may be usedto inhibit the action of such polypeptides, for example, in thetreatment of auto-immune diseases, chronic inflammatory and infectivediseases, histamine-mediated allergic reactions,prostaglandin-independent fever, bone marrow failure, silicosis,sarcoidosis, hyper-eosinophilic syndrome and lung inflammation.

[0012] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0013] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0014]FIG. 1 displays the cDNA sequence and corresponding deduced aminoacid sequence of Ckβ-4. The initial 24 amino acids represent the deducedleader sequence of Ckβ-4 such that the putative mature polypeptidecomprises 72 amino acids. The standard one-letter abbreviation for aminoacids is used.

[0015]FIG. 2 displays the cDNA sequence and corresponding deduced aminoacid sequence of Ckβ-10. The initial 23 amino acids represent theputative leader sequence of Ckβ-10 such that the putative maturepolypeptide comprises 75 amino acids. The standard one-letterabbreviation for amino acids is used.

[0016]FIG. 3 displays the amino acid sequence homology between Ckβ-4 andthe mature peptide of eotaxin (bottom).

[0017]FIG. 4 displays the amino acid sequence homology between Ckβ-10(top) and human MCP-3 (bottom).

[0018] In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which encode for themature polypeptides having the deduced amino acid sequences of FIGS. 1and 2 (SEQ ID NO:2 and 4) or for the mature polypeptide encoded by thecDNA of the clone deposited as ATCC Deposit No. 75848 (Ckβ-4) and ATCCDeposit No. 75849 Ckβ-10 on Jul. 29, 1994.

[0019] The polynucleotide encoding Ckβ-4 was discovered in a cDNAlibrary derived from a human gall bladder. Ckβ-4 is structurally relatedto the chemokine family. It contains an open reading frame encoding aprotein of 96 amino acid residues of which approximately the first 24amino acids residues are the putative leader sequence such that themature protein comprises 72 amino acids. The protein exhibits thehighest degree of homology to eotaxin with 20% identity and 37%similarity over the entire coding sequence. It is also important thatthe four spatially conserved cysteine residues in chemokines are foundin the polypeptides of the present invention.

[0020] The polynucleotide encoding Ckβ-10 was discovered in a cDNAlibrary derived from nine week early human tissue. Ckβ-10 isstructurally related to the chemokine family. It contains an openreading frame encoding a protein of 98 amino acid residues of whichapproximately the first 23 amino acids residues are the putative leadersequence such that the mature protein comprises 75 amino acids. Theprotein exhibits the highest degree of homology to MCP-3 with 65%identity and 77% similarity over the entire coding sequence.

[0021] The polynucleotides of the present invention may be in the formof RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptides may beidentical to the coding sequence shown in FIGS. 1 and 2 or that of thedeposited clones or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptides as the DNA of FIGS. 1 and 2or the deposited cDNAs.

[0022] The polynucleotides which encodes for the mature polypeptides ofFIGS. 1 and 2 or for the mature polypeptides encoded by the depositedcDNA may include: only the coding sequence for the mature polypeptide;the coding sequence for the mature polypeptide and additional codingsequence such as a leader or secretory sequence or a proproteinsequence; the coding sequence for the mature polypeptide (and optionallyadditional coding sequence) and non-coding sequence, such as introns ornon-coding sequence 5′ and/or 3′ of the coding sequence for the maturepolypeptides.

[0023] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0024] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIGS. 1 and 2 or the polypeptide encoded by the cDNA of thedeposited clones. The variant of the polynucleotides may be a naturallyoccurring allelic variant of the polynucleotides or a non-naturallyoccurring variant of the polynucleotides.

[0025] Thus, the present invention includes polynucleotides encoding thesame mature polypeptides as shown in FIGS. 1 and 2 or the same maturepolypeptides encoded by the cDNA of the deposited clones as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptides of FIGS. 1 and 2 or thepolypeptides encoded by the cDNA of the deposited clones. Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants.

[0026] As hereinabove indicated, the polynucleotides may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIGS. 1 and 2 or of the coding sequence of thedeposited clones. As known in the art, an allelic variant is analternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

[0027] The present invention also includes polynucleotides, wherein thecoding sequence for the mature polypeptides may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

[0028] Thus, for example, the polynucleotide of the present inventionmay encode for a mature protein, or for a protein having a prosequenceor for a protein having both a prosequence and a presequence (leadersequence).

[0029] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptides of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptides fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0030] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0031] Fragments of the full length gene of the present invention may beused as a hybridization probe for a cDNA library to isolate the fulllength cDNA and to isolate other cDNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 30 bases and may contain, for example, 50or more bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or MRNA to determinewhich members of the library the probe hybridizes to.

[0032] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least90%, and more preferably at least 95% identity between the sequences.The present invention particularly relates to polynucleotides whichhybridize under stringent conditions to the hereinabove-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences. The polynucleotides whichhybridize to the hereinabove described polynucleotides in a preferredembodiment encode polypeptides which either retain substantially thesame biological function or activity as the mature polypeptide encodedby the cDNAs of FIG. 1 and 2 (SEQ ID NO:1 and 3) or the depositedcDNA(s).

[0033] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO:1, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

[0034] Thus, the present invention is directed to polynucleotides havingat least a 90% and more preferably at least a 95% identity to apolynucleotide which encodes the polypeptide of SEQ ID NO:2 and 4 aswell as fragments thereof, which fragments have at least 30 bases andpreferably at least 50 bases and to polypeptides encoded by suchpolynucleotides.

[0035] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0036] The present invention further relates to chemokine polypeptideswhich have the deduced amino acid sequences of FIGS. 1 and 2 or whichhas the amino acid sequence encoded by the deposited cDNA, as well asfragments, analogs and derivatives of such polypeptides.

[0037] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptides of FIGS. 1 and 2 or that encoded by the deposited cDNA,means polypeptides which retain essentially the same biological functionor activity as such polypeptides. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

[0038] The chemokine polypeptides of the present invention may berecombinant polypeptides, natural polypeptides or a syntheticpolypeptides, preferably recombinant polypeptides.

[0039] The fragment, derivative or analog of the polypeptides of FIGS. 1and 2 or that encoded by the deposited cDNA may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0040] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0041] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0042] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 and 4 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and 4 and more preferably atleast 90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and 4 and still more preferably at least 95%similarity (still more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and 4 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0043] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0044] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0045] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0046] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the Ckβ-4 and Ckβ-10 genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

[0047] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0048] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0049] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0050] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0051] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0052] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0053] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, PBPV, PMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0054] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL andtrp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0055] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation. (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0056] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0057] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0058] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0059] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0060] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0061] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0062] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0063] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0064] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0065] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0066] The chemokine polypeptides can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0067] The chemokine polypeptides of the present invention may be anaturally purified product, or a product of chemical syntheticprocedures, or produced by recombinant techniques from a prokaryotic oreukaryotic host (for example, by bacterial, yeast, higher plant, insectand mammalian cells in culture). Depending upon the host employed in arecombinant production procedure, the polypeptides of the presentinvention may be glycosylated or may be non-glycosylated. Polypeptidesof the invention may also include an initial methionine amino acidresidue.

[0068] The chemokine polypeptides may be used to inhibit bone marrowstem cell colony formation as adjunct protective treatment during cancerchemotherapy and for leukemia.

[0069] The chemokine polypeptides may also be used to inhibit epidermalkeratinocyte proliferation for treatment of psoriasis, which ischaracterized by keratinocyte hyper-proliferation.

[0070] The chemokine polypeptides may also be used to treat solid tumorsby stimulating the invasion and activation of host defense cells, e.g.,cytotoxic T cells and macrophages. They may also be used to enhance hostdefenses against resistant chronic infections, for example,mycobacterial infections via the attraction and activation ofmicrobicidal leukocytes.

[0071] The chemokine polypeptides may also be used to treat auto-immunedisease and lymphocytic leukemias by inhibiting T cell proliferation bythe inhibition of IL2 biosynthesis.

[0072] Ckβ-4 and Ckβ-10 may also be used in wound healing, both via therecruitment of debris clearing and connective tissue promotinginflammatory cells and also via its control of excessive TGFβ-mediatedfibrosis. In this same manner, Ckβ-4 and Ckβ-10 may also be used totreat other fibrotic disorders, including liver cirrhosis,osteoarthritis and pulmonary fibrosis. The chemokine polypeptides alsoincrease the presence of eosinophils which have the distinctive functionof killing the larvae of parasites that invade tissues, as inschistosomiasis, trichinosis and ascariasis. They may also be used toregulate hematopoiesis, by regulating the activation and differentiationof various hematopoietic progenitor cells .

[0073] Chemokines may also be employed as inhibitors of angiogenesis,therefore, they have anti-tumor effects.

[0074] This invention provides a method for identification of thereceptor for the polypeptides of the present invention. The geneencoding the receptors can be identified by expression cloning. Briefly,polyadenylated RNA is prepared from a cell responsive to the polypeptideof the present invention and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptide of the present invention.Transfected cells which are grown on glass slides are exposed to labeledpolypeptides of the present invention. The polypeptides can be labeledby a variety of means including iodidation or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andretransfected using an iterative sub-pooling and rescreening process,eventually yielding a single clone that encodes the putative receptor.As an alternative approach for receptor identification, labeled ligandcan be photoaffinity linked with cell membrane or extract preparationsthat express the receptor molecule. Cross-linked material is resolved byPAGE and exposed to x-ray film. The labeled complex containing thepolypeptide-receptor can be excised, resolved into peptide fragments,and subjected to protein microsequencing. The amino acid sequenceobtained from microsequencing would be used to design a set of generateoligonucleotide probes to screen a cDNA library to identify the geneencoding the putative receptor.

[0075] This invention also provides a method of screening compounds toidentify those which bind to the receptor and elicit a second messengerresponse (agonists) or do not elicit a second messenger response(antagonists). As an example, a mammalian cell or membrane preparationexpressing the receptor would be incubated with a labeled compound. Theresponse of a known second messenger system following interaction of thecompound and the receptor is then measured. Such second messengersystems include but are not limited to, cAMP guanylate cyclase, ionchannels or phosphoinositide hydrolysis.

[0076] Potential antagonists include antibodies, or in some cases,oligonucleotides, which bind to the polypeptides. Another example of apotential antagonist is a negative dominant mutant of the polypeptides.Negative dominant mutants are polypeptides which bind to the receptor ofthe wild-type polypeptide, but fail to retain biological activity.

[0077] An assay to detect negative dominant mutants of the polypeptidesinclude an in vitro chemotaxis assay wherein a multiwell chemotaxischamber equipped with polyvinylpyrrolidone-free polycarbonate membranesis used to measure the chemoattractant ability of the polypeptides forleukocytes in the presence and absence of potential antagonist oragonist molecules.

[0078] Antisense constructs prepared using antisense technology are alsopotential antagonists. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.For example, the 5′ coding portion of the polynucleotide sequence, whichencodes for the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple-helix, see Leeet al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456(1988); and Dervan et al., Science, 251: 1360 (1991)), therebypreventing transcription and the production of the polypeptides. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the MRNA molecule into the polypeptides (antisense—Okano,J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the polypeptides.

[0079] Another potential antagonist is a peptide derivative of thepolypeptides which are naturally or synthetically modified analogs ofthe polypeptides that have lost biological function yet still recognizeand bind to the receptors of the polypeptides to thereby effectivelyblock the receptors. Examples of peptide derivatives include, but arenot limited to, small peptides or peptide-like molecules.

[0080] The antagonists may be employed to inhibit the chemotaxis andactivation of macrophages and their precursors, and of neutrophils,basophils, B lymphocytes and some T cell subsets, e.g., activated andCD8 cytotoxic T cells and natural killer cells, in auto-immune andchronic inflammatory and infective diseases. Examples of auto-immunediseases include rheumatoid arthritis, multiple sclerosis, andinsulin-dependent diabetes. Some infectious diseases include silicosis,sarcoidosis, idiopathic pulmonary fibrosis by preventing the recruitmentand activation of mononuclear phagocytes, idiopathic hyper-eosinophilicsyndrome by preventing eosinophil production and migration, endotoxicshock by preventing the migration of macrophages and their production ofthe chemokine polypeptides of the present invention.

[0081] The antagonists may also be employed for treatingatherosclerosis, by preventing monocyte infiltration in the artery wall.

[0082] The antagonists may also be employed to treat histamine-mediatedallergic reactions by inhibiting chemokine-induced mast cell andbasophil degranulation and release of histamine.

[0083] The antagonists may also be employed to treat inflammation bypreventing the attraction of monocytes to a wound area. They may also beemployed to regulate normal pulmonary macrophage populations, sinceacute and chronic inflammatory pulmonary diseases are associated withsequestration of mononuclear phagocytes in the lung.

[0084] Antagonists may also be employed to treat rheumatoid arthritis bypreventing the attraction of monocytes into synovial fluid in the jointsof patients. Monocyte influx and activation plays a significant role inthe pathogenesis of both degenerative and inflammatory arthropathies.

[0085] The antagonists may be employed to interfere with the deleteriouscascades attributed primarily to IL-1 and TNF, which prevents thebiosynthesis of other inflammatory cytokines. In this way, theantagonists may be employed to prevent inflammation. The antagonists mayalso be employed to inhibit prostaglandin-independent fever induced bychemokines.

[0086] The antagonists may also be employed to treat cases of bonemarrow failure, for example, aplastic anemia and myelodysplasticsyndrome.

[0087] The antagonists may also be employed to treat asthma and allergyby preventing eosinophil accumulation in the lung. The antagonists maybe employed in a composition with a pharmaceutically acceptable carrier,e.g., as hereinafter described.

[0088] The chemokine polypeptides and agonists or antagonists of thepresent invention may be employed in combination with a suitablepharmaceutical carrier. Such compositions comprise a therapeuticallyeffective amount of the polypeptide, agonist or antagonist, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

[0089] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides, agonists and antagonists of the presentinvention may be employed in conjunction with other therapeuticcompounds.

[0090] The pharmaceutical compositions may be administered in aconvenient manner such as by the topical, intravenous, intraperitoneal,intramuscular, intratumor, subcutaneous, intranasal or intradermalroutes. The polypeptides are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, the polypeptides will be administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

[0091] The chemokine polypeptides and agonists or antagonists which arepolypeptides may be employed in accordance with the present invention byexpression of such polypeptides in vivo, which is often referred to as“gene therapy.”

[0092] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0093] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0094] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0095] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0096] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide. The retroviral plasmid vector isemployed to transduce packaging cell lines to form producer cell lines.Examples of packaging cells which may be transfected include, but arenot limited to, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2,ψCRE, ψCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described inMiller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which isincorporated herein by reference in its entirety. The vector maytransduce the packaging cells through any means known in the art. Suchmeans include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

[0097] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0098] This invention is also related to the use of the gene of thepresent invention as a diagnostic. Detection of a mutated form of thegene will allow a diagnosis of a disease or a susceptibility to adisease which results from underexpression of the polypeptides of thepresent invention.

[0099] Individuals carrying mutations in the gene of the presentinvention may be detected at the DNA level by a variety of techniques.Nucleic acids for diagnosis may be obtained from a patient's cells,including but not limited to blood, urine, saliva, tissue biopsy andautopsy material. The genomic DNA may be used directly for detection ormay be amplified enzymatically by using PCR (Saiki et al., Nature,324:163-166 (1986)) prior to analysis. RNA or cDNA may also be used forthe same purpose. As an example, PCR primers complementary to thenucleic acid encoding the polypeptides of the present invention can beused to identify and analyze mutations. For example, deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to radiolabeled RNA or alternatively,radiolabeled antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

[0100] Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

[0101] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0102] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0103] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0104] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0105] The present invention also relates to a diagnostic assay fordetecting altered levels of the polypeptide of the present invention invarious tissues since an over-expression of the proteins compared tonormal control tissue samples can detect the presence of disorders ofthe host. Assays used to detect levels of the polypeptide of the presentinvention in a sample derived from a host are well-known to those ofskill in the art and include radioimmunoassays, competitive-bindingassays, Western Blot analysis and preferably an ELISA assay. An ELISAassay initially comprises preparing an antibody specific to a Ckβ-4 or10 antigen, preferably a monoclonal antibody. In addition a reporterantibody is prepared against the monoclonal antibody. To the reporterantibody is attached a detectable reagent such as radioactivity,fluorescence or in this example a horseradish peroxidase enzyme. Asample is now removed from a host and incubated on a solid support, e.g.a polystyrene dish, that binds the proteins in the sample. Any freeprotein binding sites on the dish are then covered by incubating with anon-specific protein such as bovine serum albumin. Next, the monoclonalantibody is incubated in the dish during which time the monoclonalantibodies attached to the polypeptide of the present invention attachedto the polystyrene dish. All unbound monoclonal antibody is washed outwith buffer. The reporter antibody linked to horseradish peroxidase isnow placed in the dish resulting in binding of the reporter antibody toany monoclonal antibody bound to the polypeptide of the presentinvention. Unattached reporter antibody is then washed out. Peroxidasesubstrates are then added to the dish and the amount of color developedin a given time period is a measurement of the amount of the polypeptideof the present invention present in a given volume of patient samplewhen compared against a standard curve.

[0106] A competition assay may be employed wherein antibodies specificto the polypeptide of the present invention are attached to a solidsupport and a labeled polypeptide of the present invention and a samplederived from the host are passed over the solid support and the amountof label detected attached to the solid support can be correlated to aquantity of the polypeptide of the present invention in the sample.

[0107] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0108] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0109] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0110] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAhaving at least 50 or 60 bases. For a review of this technique, seeVerma et al., Human Chromosomes: a Manual of Basic Techniques, PergamonPress, New York (1988).

[0111] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0112] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0113] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0114] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0115] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0116] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0117] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0118] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0119] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0120] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0121] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0122] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0123] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0124] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units to T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0125] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1

[0126] Bacterial Expression and Purification of Ckβ-4

[0127] The DNA sequence encoding for Ckβ-4, ATCC # 75848, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the processed Ckβ-4 protein (minus the putative signalpeptide sequence). Additional nucleotides corresponding to Ckβ-4 wereadded to the 5′ and 3′ sequences respectively. The 5′ oligonucleotideprimer has the sequence 5′ CCCGCATGCAAGCAGCAAGCAACTTT 3′ (SEQ ID NO:5)contains a SphI restriction enzyme site (bold) followed by 17nucleotides of Ckβ-4 coding sequence (underlined) starting from thesecond nucleotide of the sequences coding for the mature protein. TheATG codon is included in the SphI site. In the next codon following theATG, the first base is from the SphI site and the remaining two basescorrespond to the second and third base of the first codon of theputative mature protein. As a consequence, the first base in this codonis changed from G to C compared with the original sequences, resultingin an E to Q substitution in the recombinant protein. The 3′ sequence,5′ AAAGGATCCCATGTTCTTGACTTTTTTACT 3′ (SEQ ID NO:6) containscomplementary sequences to a BamH1 site (bold) and is followed by 21nucleotides of gene specific sequences preceding the termination codon.The restriction enzyme sites correspond to the restriction enzyme siteson the bacterial expression vector pQE-70 (Qiagen, Inc. 9259 EtonAvenue, Chatsworth, CA, 91311). pQE-70 encodes antibiotic resistance(Amp^(r)), a bacterial origin of replication (ori), an IPTG-regulatablepromoter operator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-70 was then digested with SphI and BamH1.The amplified sequences were ligated into pQE-70 and were inserted inframe with the sequence encoding for the histidine tag and the RBS. FIG.8 shows a schematic representation of this arrangement. The ligationmixture was then used to transform the E. coli strain available fromQiagen under the trademark M15/rep 4 by the procedure described inSambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989). M15/rep4 contains multiple copies of theplasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(r)). Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA was isolated and confirmed by restrictionanalysis. Clones containing the desired constructs were grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells were grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells were grownan extra 3 to 4 hours. Cells were then harvested by centrifugation. Thecell pellet was solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized Ckβ-4 was purified from thissolution by chromatography on a Nickel-Chelate column under conditionsthat allow for tight binding by proteins containing the 6-His tag(Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). Ckβ-4 (>98%pure) was eluted from the column in 6 molar guanidine HCl pH 5.0.Protein renaturation out of GnHCl can be accomplished by severalprotocols (Jaenicke, R. and Rudolph, R., Protein Structure—A PracticalApproach, IRL Press, New York (1990)). Initially, step dialysis isutilized to remove the GnHCL. Alternatively, the purified proteinisolated from the Ni-chelate column can be bound to a second column overwhich a decreasing linear GnHCL gradient is run. The protein is allowedto renature while bound to the column and is subsequently eluted with abuffer containing 250 mM Imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5and 10% Glycerol. Finally, soluble protein is dialyzed against a storagebuffer containing 5 mM Ammonium Bicarbonate.

EXAMPLE 2

[0128] Bacterial Expression and Purification of Ckβ-10

[0129] The DNA sequence encoding for Ckβ-10, ATCC # 75849, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the processed Ckβ-10 protein (minus the signal peptidesequence) and the vector sequences 3′ to the Ckβ-10 gene. Additionalnucleotides corresponding to Ckβ-10 were added to the 5′ and 3′sequences respectively. The 5′ oligonucleotide primer has the sequence5° CCCGCATGCAGCCAGATGCACTCAACG 3′ (SEQ ID NO:7) contains a SphIrestriction enzyme site (bold) followed by 19 nucleotides of Ckβ-10coding sequence (underlined) starting from the sequences coding for themature protein. The ATG codon is included in the SphI site. The 3′sequence, 5′ AAAGGATCCAGTCTTCAGGGTGTGAGCT 3′ (SEQ ID NO:8) containscomplementary sequences to a BamH1 site (bold) and is followed by 19nucleotides of gene specific sequences preceding the termination codon.The restriction enzyme sites correspond to the restriction enzyme siteson the bacterial expression vector pQE-70 (Qiagen, Inc. 9259 EtonAvenue, Chatsworth, Calif., 91311). pQE-70 encodes antibiotic resistance(Amp^(r)), a bacterial origin of replication (ori), an IPTG-regulatablepromoter operator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-70 was then digested with SphI and BamH1.The amplified sequences were ligated into pQE-70 and were inserted inframe with the sequence encoding for the histidine tag and the RBS. FIG.10 shows a schematic representation of this arrangement. The ligationmixture was then used to transform the E. coli strain available fromQiagen under the trademark M15/rep 4 by the procedure described inSambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989). M15/rep4 contains multiple copies of theplasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(r)). Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA was isolated and confirmed by restrictionanalysis. Clones containing the desired constructs were grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells were grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells were grownan extra 3 to 4 hours. Cells were then harvested by centrifugation. Thecell pellet was solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized Ckβ-10 was purified from thissolution by chromatography on a Nickel-Chelate column under conditionsthat allow for tight binding by proteins containing the 6-His tag(Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). Ckβ-10 (>98%pure) was eluted from the column in 6 molar guanidine HCl pH 5.0.Protein renaturation out of GnHCl can be accomplished by severalprotocols (Jaenicke, R. and Rudolph, R., Protein Structure—A PracticalApproach, IRL Press, New York (1990)). Initially, step dialysis isutilized to remove the GnHCL. Alternatively, the purified proteinisolated from the Ni-chelate column can be bound to a second column overwhich a decreasing linear GnHCL gradient is run. The protein is allowedto renature while bound to the column and is subsequently eluted with abuffer containing 250 mM Imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5and 10% Glycerol. Finally, soluble protein is dialyzed against a storagebuffer containing 5 mM Ammonium Bicarbonate. The protein was thenanalyzed on an SDS-PAGE gel

EXAMPLE 3

[0130] Expression of Recombinant Ckβ-4 in COS cells

[0131] The expression of plasmid, Ckβ-4 HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire Ckβ-4 precursorand a HA tag fused in frame to its 3′ end was cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter. The HA tag correspond to an epitopederived from the influenza hemagglutinin protein as previously described(I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.Lerner, 1984, Cell 37, 767). The infusion of HA tag to the targetprotein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0132] The plasmid construction strategy is described as follows:

[0133] The DNA sequence encoding for Ckβ-4, ATTC. # 75848 wasconstructed by PCR on the original EST cloned using two primers: the 5′primer 5′ GGAAAGCTTATGTGCTGTACCAAGAGTTT 3′ (SEQ ID NO:9) contains aHindIII site followed by 20 nucleotides of Ckβ-4 coding sequencestarting from the initiation codon; the 3′ sequence 5′CGCTCTAGATTAAGCGTAGTCTGGGACGTCGTATGGGTAACATGGTTCCTTGACTTTTT 3′ (SEQ IDNO:10) contains complementary sequences to XbaI site, translation stopcodon, HA tag and the last 20 nucleotides of the Ckβ-4 coding sequence(not including the stop codon). Therefore, the PCR product contains aHindIII site, Ckβ-4 coding sequence followed by HA tag fused in frame, atranslation termination stop codon next to the HA tag, and an XbaI site.The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digestedwith HindIII and XbaI restriction enzyme and ligated. The ligationmixture was transformed into E. coli strain SURE (available fromStratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla,Calif. 92037) the transformed culture was plated on ampicillin mediaplates and resistant colonies were selected. Plasmid DNA was isolatedfrom transformants and examined by restriction analysis for the presenceof the correct fragment. For expression of the recombinant Ckβ-4, COScells were transfected with the expression vector by DEAE-DEXTRANmethod. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989)). The expressionof the Ckβ-4 HA protein was detected by radiolabelling andimmunoprecipitation method. (E. Harlow, D. Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cellswere labelled for 8 hours with ³⁵S-cysteine two days post transfection.Culture media were then collected and cells were lysed with detergent(RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mMTris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)). both cell lysateand culture media were precipitated with a HA specific monoclonalantibody. Proteins precipitated were analyzed by SDS-PAGE.

EXAMPLE 4

[0134] Expression of Recombinant Ckβ-10 in COS cells

[0135] The expression of plasmid, Ckβ-10 HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire Ckβ-10precursor and a HA tag fused in frame to its 3′ end was cloned into thepolylinker region of the vector, therefore, the recombinant proteinexpression is directed under the CMV promoter. The HA tag correspond toan epitope derived from the influenza hemagglutinin protein aspreviously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M.Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag tothe target protein allows easy detection of the recombinant protein withan antibody that recognizes the HA epitope.

[0136] The plasmid construction strategy is described as follows:

[0137] The DNA sequence encoding for Ckβ-10, ATTC. # 75849, wasconstructed by PCR on the original EST cloned using two primers: the 5′primer 5′ GGAAAGCTTATGAAAGTTTCTGCAGTGC 3′ (SEQ ID NO:1l) contains aHindIII site followed by 19 nucleotides of Ckβ-10 coding sequencestarting from the initiation codon; the 3′ sequence 5′CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAAGTCTTCAGGGTGTGAGCT 3′ (SEQ IDNO:12) contains complementary sequences to XbaI site, translation stopcodon, HA tag and the last 19 nucleotides of the Ckβ-10 coding sequence(not including the stop codon). Therefore, the PCR product contains aHindIII site, Ckβ-10 coding sequence followed by HA tag fused in frame,a translation termination stop codon next to the HA tag, and an XbaIsite. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, weredigested with HindIII and BamHI restriction enzyme and ligated. Theligation mixture was transformed into E. coli strain SURE (availablefrom Stratagene Cloning Systems, 11099 North Torrey Pines Road, LaJolla, Calif. 92037) the transformed culture was plated on ampicillinmedia plates and resistant colonies were selected. Plasmid DNA wasisolated from transformants and examined by restriction analysis for thepresence of the correct fragment. For expression of the recombinantCkβ-10, COS cells were transfected with the expression vector byDEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989) ).The expression of the Ckβ-10 HA protein was detected by radiolabellingand immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cellswere labelled for 8 hours with ³⁵S-cysteine two days post transfection.Culture media were then collected and cells were lysed with detergent(RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mMTris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysateand culture media were precipitated with a HA specific monoclonalantibody. Proteins precipitated were analyzed by SDS-PAGE.

EXAMPLE 5

[0138] Cloning and Expression of Ckβ-10 using the baculovirus ExpressionSystem

[0139] The DNA sequence encoding the full length Ckβ-10 oligonucleotideprimers corresponding to the 5′ and 3′ sequences of the gene:

[0140] The 5′ primer has the sequence 5′ CGCGGGATCCTTAACCTTCAACATGAAA(SEQ ID NO:13) and contains a BamHI restriction enzyme site (in bold)followed by 12 nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (J. Mol. Biol. 1987, 196,947-950, Kozak, M.), and just behind, is the first 6 nucleotides of theCkβ-10 coding sequence (the initiation codon for translation “ATG” isunderlined).

[0141] The 3′ primer has the sequence 5′ CGCGGGTACCTTAACACATAGTACATTTT(SEQ ID NO:14) and contains the cleavage site for the restrictionendonuclease Asp781 and 19 nucleotides complementary to the 3′non-translated sequence of the Ckβ-10 gene. The amplified sequences wereisolated from a 1% agarose gel using a commercially available kit(“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment was thendigested with the endonucleases BamHI and Asp781 and then purified againon a 1% agarose gel. This fragment is designated F2.

[0142] The vector PRG1 (modification of pVL941 vector, discussed below)is used for the expression of the Ckβ-10 protein using the baculovirusexpression system (for review see: Summers, M. D and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).promoter of the Autographa californica nuclear polyhedrosis virus(AcMNPV) followed by the recognition sites for the restrictionendonucleases BamHI and Asp781. The polyadenylation site of the simianvirus (SV)40 is used for efficient polyadenylation. For an easyselection of recombinant viruses the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoterfollowed by the polyadenylation signal of the polyhedrin gene. Thepolyhedrin sequences are flanked at both sides by viral sequences forthe cell-mediated homologous recombination of cotransfected wild-typeviral DNA. Many other baculovirus vectors could be used in place of pRG1such as pAc373, pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D.,Virology, 170:31-39).

[0143] The plasmid was digested with the restriction enzymes BamHI andAsp781 and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA was then isolated from a 1% agarosegel. This vector DNA is designated V2.

[0144] Fragment F2 and the dephosphorylated plasmid V2 were ligated withT4 DNA ligase. E. coli HB101 cells were then transformed and bacteriaidentified that contained the plasmid (pBacCkβ-10) with the Ckβ-10 geneusing the enzymes BamHI and Asp781. The sequence of the cloned fragmentwas confirmed by DNA sequencing.

[0145] 5 μg of the plasmid pBacCkβ-10 were cotransfected with 1.0 βg ofa commercially available linearized baculovirus (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofectionmethod (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

[0146] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacCkβ-10were mixed in a sterile well of a microtiter plate containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added dropwise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace' medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27° C. After 5hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at27° C for four days.

[0147] After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0148] Four days after the serial dilution of the viruses was added tothe cells, blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculoviruses was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

[0149] Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-Ckβ-10 at a multiplicity of infection (MOI) of 2. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 6

[0150] Expression via Gene Therapy

[0151] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks. pMV-7 (Kirschmeier, P. T. etal, DNA, 7:219-25 (1988) flanked by the long terminal repeats of theMoloney murine sarcoma virus, is digested with EcoRI and HindIII andsubsequently treated with calf intestinal phosphatase. The linear vectoris fractionated on agarose gel and purified, using glass beads.

[0152] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer further includes a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the amplified EcoRI andHindIII fragment are added together, in the presence of T4 DNA ligase.The resulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

[0153] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells.

[0154] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0155] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0156] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 20 291 base pairs nucleic acid single linear DNA (genomic) CDS 1..2881 ATG TGC TGT ACC AAG AGT TTG CTC CTG GCT GCT TTG ATG TCA GTG CTG 48 MetCys Cys Thr Lys Ser Leu Leu Leu Ala Ala Leu Met Ser Val Leu 1 5 10 15CTA CTC CAC CTC TGC GGC GAA TCA GAA GCA GCA AGC AAC TTT GAC TGC 96 LeuLeu His Leu Cys Gly Glu Ser Glu Ala Ala Ser Asn Phe Asp Cys 20 25 30 TGTCTT GGA TAC ACA GAC CGT ATT CTT CAT CCT AAA TTT ATT GTG GGC 144 Cys LeuGly Tyr Thr Asp Arg Ile Leu His Pro Lys Phe Ile Val Gly 35 40 45 TTC ACACGG CAG CTG GCC AAT GAA GGC TGT GAC ATC AAT GCT ATC ATC 192 Phe Thr ArgGln Leu Ala Asn Glu Gly Cys Asp Ile Asn Ala Ile Ile 50 55 60 TTT CAC ACAAAG AAA AAG TTG TCT GTG TGC GCA AAT CCA AAA CAG ACT 240 Phe His Thr LysLys Lys Leu Ser Val Cys Ala Asn Pro Lys Gln Thr 65 70 75 80 TGG GTG AAATAT ATT GTG CGT CTC CTC AGT AAA AAA GTC AAG AAC ATG 288 Trp Val Lys TyrIle Val Arg Leu Leu Ser Lys Lys Val Lys Asn Met 85 90 95 TAA 291 96amino acids amino acid linear protein 2 Met Cys Cys Thr Lys Ser Leu LeuLeu Ala Ala Leu Met Ser Val Leu 1 5 10 15 Leu Leu His Leu Cys Gly GluSer Glu Ala Ala Ser Asn Phe Asp Cys 20 25 30 Cys Leu Gly Tyr Thr Asp ArgIle Leu His Pro Lys Phe Ile Val Gly 35 40 45 Phe Thr Arg Gln Leu Ala AsnGlu Gly Cys Asp Ile Asn Ala Ile Ile 50 55 60 Phe His Thr Lys Lys Lys LeuSer Val Cys Ala Asn Pro Lys Gln Thr 65 70 75 80 Trp Val Lys Tyr Ile ValArg Leu Leu Ser Lys Lys Val Lys Asn Met 85 90 95 297 base pairs nucleicacid single linear DNA (genomic) CDS 1..294 3 ATG AAA GTT TCT GCA GTGCTT CTG TGC CTG CTG CTC ATG ACA GCA GCT 48 Met Lys Val Ser Ala Val LeuLeu Cys Leu Leu Leu Met Thr Ala Ala 1 5 10 15 TTC AAC CCC CAG GGA CTTGCT CAG CCA GAT GCA CTC AAC GTC CCA TCT 96 Phe Asn Pro Gln Gly Leu AlaGln Pro Asp Ala Leu Asn Val Pro Ser 20 25 30 ACT TGC TGC TTC ACA TTT AGCAGT AAG AAG ATC TCC TTG CAG AGG CTG 144 Thr Cys Cys Phe Thr Phe Ser SerLys Lys Ile Ser Leu Gln Arg Leu 35 40 45 AAG AGC TAT GTG ATC ACC ACC AGCAGG TGT CCC CAG AAG GCT GTC ATC 192 Lys Ser Tyr Val Ile Thr Thr Ser ArgCys Pro Gln Lys Ala Val Ile 50 55 60 TTC AGA ACC AAA CTG GGC AAG GAG ATCTGT GCT GAC CCA AAG GAG AAG 240 Phe Arg Thr Lys Leu Gly Lys Glu Ile CysAla Asp Pro Lys Glu Lys 65 70 75 80 TGG GTC CAG AAT TAT ATG AAA CAC CTGGGC CGG AAA GCT CAC ACC CTG 288 Trp Val Gln Asn Tyr Met Lys His Leu GlyArg Lys Ala His Thr Leu 85 90 95 AAG ACT TGA 297 Lys Thr 98 amino acidsamino acid linear protein 4 Met Lys Val Ser Ala Val Leu Leu Cys Leu LeuLeu Met Thr Ala Ala 1 5 10 15 Phe Asn Pro Gln Gly Leu Ala Gln Pro AspAla Leu Asn Val Pro Ser 20 25 30 Thr Cys Cys Phe Thr Phe Ser Ser Lys LysIle Ser Leu Gln Arg Leu 35 40 45 Lys Ser Tyr Val Ile Thr Thr Ser Arg CysPro Gln Lys Ala Val Ile 50 55 60 Phe Arg Thr Lys Leu Gly Lys Glu Ile CysAla Asp Pro Lys Glu Lys 65 70 75 80 Trp Val Gln Asn Tyr Met Lys His LeuGly Arg Lys Ala His Thr Leu 85 90 95 Lys Thr 26 base pairs nucleic acidsingle linear DNA (genomic) 5 CCCGCATGCA AGCAGCAAGC AACTTT 26 30 basepairs nucleic acid single linear DNA (genomic) 6 AAAGGATCCC ATGTTCTTGACTTTTTTACT 30 27 base pairs nucleic acid single linear DNA (genomic) 7CCCGCATGCA GCCAGATGCA CTCAACG 27 28 base pairs nucleic acid singlelinear DNA (genomic) 8 AAAGGATCCA GTCTTCAGGG TGTGAGCT 28 29 base pairsnucleic acid single linear DNA (genomic) 9 GGAAAGCTTA TGTGCTGTACCAAGAGTTT 29 59 base pairs nucleic acid single linear DNA (genomic) 10CGCTCTAGAT TAAGCGTAGT CTGGGACGTC GTATGGGTAA CATGGTTCCT TGACTTTTT 59 28base pairs nucleic acid single linear DNA (genomic) 11 GGAAAGCTTATGAAAGTTTC TGCAGTGC 28 58 base pairs nucleic acid single linear DNA(genomic) 12 CGCTCTAGAT CAAGCGTAGT CTGGGACGTC GTATGGGTAA GTCTTCAGGGTGTGAGCT 58 28 base pairs nucleic acid single linear DNA (genomic) 13CGCGGGATCC TTAACCTTCA ACATGAAA 28 29 base pairs nucleic acid singlelinear DNA (genomic) 14 CGCGGGTACC TTAACACATA GTACATTTT 29 27 base pairsnucleic acid single linear DNA (genomic) 15 GCGGGATCCT TAACCTTCAACATGAAA 27 29 base pairs nucleic acid single linear DNA (genomic) 16CGCGGGTACC TTAACACATA GTACATTTT 29 70 amino acids amino acid singlelinear protein 17 His Pro Gly Ile Pro Ser Ala Cys Cys Phe Arg Val ThrAsn Ile Cys 1 5 10 15 Lys Ile Ser Phe Gln Ala Leu Lys Ser Tyr Lys IleIle Thr Ser Ser 20 25 30 Lys Cys Pro Gln Thr Ala Ile Val Phe Glu Ile LysPro Asp Lys Met 35 40 45 Ile Cys Ala Asp Pro Arg Xaa Xaa Trp Val Gln AspAla Lys Lys Tyr 50 55 60 Leu Asp Gln Ile Ser Gln 65 70 99 amino acidsamino acid single linear protein 18 Met Lys Ala Ser Ala Ala Leu Leu CysLeu Leu Leu Thr Ala Ala Ala 1 5 10 15 Phe Ser Pro Gln Gly Leu Ala GlnPro Val Gly Ile Asn Thr Ser Thr 20 25 30 Thr Cys Cys Tyr Arg Phe Ile AsnLys Lys Ile Pro Lys Gln Arg Leu 35 40 45 Glu Ser Tyr Arg Arg Thr Thr SerSer His Cys Pro Arg Glu Ala Val 50 55 60 Ile Phe Lys Thr Lys Leu Asp LysGlu Ile Cys Ala Asp Pro Thr Gln 65 70 75 80 Lys Trp Val Gln Asp Phe MetLys His Leu Asp Lys Lys Thr Gln Thr 85 90 95 Pro Lys Leu 76 amino acidsamino acid single linear DNA (genomic) 19 Gln Pro Val Gly Ile Asn ThrSer Thr Thr Cys Cys Tyr Arg Phe Ile 1 5 10 15 Asn Lys Lys Ile Pro LysGln Arg Leu Glu Ser Tyr Arg Arg Thr Thr 20 25 30 Ser Ser His Cys Pro ArgGlu Ala Val Ile Phe Lys Thr Lys Leu Asp 35 40 45 Lys Glu Ile Cys Ala AspPro Thr Gln Lys Trp Val Gln Asp Phe Met 50 55 60 Lys His Leu Asp Lys LysThr Gln Thr Pro Lys Leu 65 70 75 74 amino acids amino acid single linearprotein 20 Gly Pro Ala Ser Val Pro Thr Thr Cys Cys Phe Asn Leu Ala AsnArg 1 5 10 15 Lys Ile Pro Leu Gln Arg Leu Glu Ser Tyr Arg Arg Ile ThrSer Gly 20 25 30 Lys Cys Pro Gln Lys Ala Val Ile Phe Lys Thr Lys Leu AlaLys Asp 35 40 45 Ile Cys Ala Asp Pro Lys Lys Lys Trp Val Gln Asp Ser MetLys Tyr 50 55 60 Leu Asp Gln Lys Ser Pro Thr Pro Lys Pro 65 70

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding thepolypeptide comprising amino acid −24 to amino acid 72 as set forth inSEQ ID NO:2; (b) a polynucleotide encoding the polypeptide comprisingamino acid 1 to amino acid 72 as set forth in SEQ ID NO:2 (c) apolynucleotide encoding the polypeptide comprising amino acid −23 toamino acid 75 as set forth in SEQ ID NO:4; (d) a polynucleotide encodingthe polypeptide comprising amino acid 1 to amino acid 75 as set forth inSEQ ID NO:4; (e) a polynucleotide capable of hybridizing to and which isat least 70% identical to the polynucleotide of (a), (b), (c) or (d);and (f) a polynucleotide fragment of the polynucleotide of (a), (b),(c), (d) or (e).
 2. The polynucleotide of claim 1 wherein thepolynucleotide is DNA.
 3. The polynucleotide of claim 2 which encodes amember selected from the group consisting of: (a) the polypeptidecomprising amino acid 1 to 72 of SEQ ID NO:2; and (b) the polypeptidecomprising amino acid 1 to 75 of SEQ ID NO:4.
 4. An isolatedpolynucleotide comprising a member selected from the group consistingof: (a) a polynucleotide which encodes a mature polypeptide having theamino acid sequence expressed by the DNA contained in ATCC Deposit No.75848; (b) a polynucleotide which encodes a mature polypeptide havingthe amino acid sequence expressed by the DNA contained in ATCC DepositNo. 75849; (c) a polynucleotide capable of hybridizing to and which isat least 70% identical to the polynucleotide of (a) or (b); and (d) apolynucleotide fragment of the polynucleotide of (a), (b) or (c).
 5. Thepolynucleotide of claim 1 comprising the sequence as set forth in SEQ IDNo. 1 from nucleotide 73 to nucleotide
 291. 6. The polynucleotide ofclaim 1 comprising the sequence as set forth in SEQ ID No. 1 fromnucleotide 70 to nucleotide
 297. 7. A vector containing the DNA of claim2.
 8. A host cell genetically engineered with the vector of claim
 7. 9.A process for producing a polypeptide comprising: expressing from thehost cell of claim 8 the polypeptide encoded by said DNA.
 10. A processfor producing cells capable of expressing a polypeptide comprisinggenetically engineering cells with the vector of claim
 7. 11. Apolypeptide encoded by the polynucleotide of claim 1 comprising a memberselected from the group consisting of; (i) a mature polypeptide havingthe deduced amino acid sequence of SEQ ID NO:2 and fragments, analogsand derivatives thereof; (ii) a mature polypeptide encoded by the cDNAof ATCC Deposit No. 75848 and fragments, analogs and derivatives of saidpolypeptide; (iii) a mature polypeptide having the deduced amino acidsequence of SEQ ID NO:4 and fragments, analogs and derivatives thereof;and (iv) a mature polypeptide encoded by the cDNA of ATCC Deposit No.75849 and fragments, analogs and derivatives of said polypeptide. 12.The polypeptide of claim 11 wherein the polypeptide comprises amino acid1 to amino acid 72 of SEQ ID NO:2.
 13. A compound which inhibitsactivation of the receptor for the polypeptide of claim
 11. 14. Acompound which activates the receptor for the polypeptide of claim 11.15. A method for the treatment of a patient having need of a polypeptideof claim 11 comprising: administering to the patient a therapeuticallyeffective amount of the polypeptide.
 16. The method of claim 15 whereinsaid therapeutically effective amount of the polypeptide is administeredby providing to the patient DNA encoding said polypeptide and expressingsaid polypeptide in vivo.
 17. A method for the treatment of a patienthaving need to inhibit a chemokine polypeptide comprising: administeringto the patient a therapeutically effective amount of the compound ofclaim
 13. 18. A process for diagnosing a disease or a susceptibility toa disease related to an under-expression of the polypeptide of claim 11comprising: determining a mutation in a nucleic acid sequence encodingsaid polypeptide.
 19. A diagnostic process comprising: analyzing for thepresence of the polypeptide of claim 11 in a sample derived from a host.20. A method for identifying agonist or antagonist compounds to thepolypeptide of claim 11 comprising: contacting a cell expressing on thace thereof a receptor for the polypeptide, said receptor beingassociated with a second component capable of providing a detectablesignal in response to the binding of a compound to said receptor, withan analytically detectable compound under conditions to permit bindingto the receptor; detecting the absence or presence of a signal generatedfrom the interaction of the compound with the receptor.